Defective survival and activation of thymocytes in transgenic mice expressing a catalytically inactive form of Ca2+/calmodulin-dependent protein kinase IV

We have generated transgenic mice that express a catalytically inactive form of Ca2+/calmodulin-dependent protein kinase IV (CaMKIV) specifically in thymic T cells. The presence of this protein results in a markedly reduced thymic cellularity, although the distribution of the remaining cells is normal based on evaluation of the CD4 and CD8 cell surface antigens that are used to gauge T cell development. Isolated thymic T cells from the transgenic mice also show a dramatically decreased survival rate when evaluated in culture under conditions that do not favor activation. When challenged with an activating stimulus such as alpha-CD3 or a combination of phorbol ester plus ionophore, the cells are severely compromised in their ability to produce the cytokine interleukin-2 (IL-2). Reduction of IL-2 production is secondary to the inability to phosphorylate the cAMP response element binding protein, CREB, and induce expression of the immediate early genes such as Fos B that are required to transactivate the IL-2 promoter. Because transgene expression was regulated by the proximal promoter of the murine lck gene and this promoter is inactivated in T cells that exit the thymus, the mutant hCaMKIV is not present in peripheral T cells. Consequently, T lymphocytes present in the spleen can be activated normally in response to either stimulus mentioned above, demonstrating that the effects of the inactive CaMKIV on activation are reversible. Our results suggest that CaMKIV may represent a physiologically relevant CREB kinase in T cells and that the enzyme is also required to ensure normal expansion of T cells in the thymus. Whereas the pathway responsible for this latter role is yet to be elucidated, it is unlikely to include CREB phosphorylation

Modulation of Opiate-Related Signaling Molecules in Morphine-Dependent Conditioned Behavior: Conditioned Place Preference to Morphine Induces CREB Phosphorylation

Opiate addiction is a chronic, relapsing behavioral disorder where learned associations that develop between the abused opiate and the environment in which it is consumed are brought about through Pavlovian (classical) conditioning processes. However, the signaling mechanisms/pathways regulating the mechanisms that underlie the responses to opiate-associated cues or the development of sensitization as a consequence of repeated context-independent administration of opiates are unknown. In this study we examined the phosphorylation levels of various classic signaling molecules in brain regions implicated in addictive behaviors after acute and repeated morphine administration. An unbiased place conditioning protocol was used to examine changes in phosphorylation that are associated with (1) the expression of the rewarding effects of morphine and (2) the sensitization that develops to this effect. We also examined the effects of a δ-receptor antagonist on morphine-induced conditioned behavior and on the phosphorylation of classic signaling molecules in view of data showing that blockade of δ-opioid receptor (δOR) prevents the development of sensitization to the rewarding effects of morphine. We find that CREB phosphorylation is specifically induced upon the expression of a sensitized response to morphine-induced conditioned behavior in brain areas related to memory consolidation, such as the hippocampus and cortex. A similar effect is also observed, albeit to a lesser extent, in the case of the GluR1 subunit of AMPA glutamate receptor. These increases in the phosphorylation levels of CREB and pGluR1 are significantly blocked by pretreatment with a δOR antagonist. These results indicate a critical role for phospho-CREB, AMPA, and δOR activities in mediating the expression of a sensitized response to morphine-dependent conditioned behavior

Altered mGluR5-Homer scaffolds and corticostriatal connectivity in a Shank3 complete knockout model of autism

Human neuroimaging studies suggest that aberrant neural connectivity underlies behavioural deficits in autism spectrum disorders (ASDs), but the molecular and neural circuit mechanisms underlying ASDs remain elusive. Here, we describe a complete knockout mouse model of the autism-associated Shank3 gene, with a deletion of exons 4-22 (Δe4-22). Both mGluR5-Homer scaffolds and mGluR5-mediated signalling are selectively altered in striatal neurons. These changes are associated with perturbed function at striatal synapses, abnormal brain morphology, aberrant structural connectivity and ASD-like behaviour. In vivo recording reveals that the cortico-striatal-thalamic circuit is tonically hyperactive in mutants, but becomes hypoactive during social behaviour. Manipulation of mGluR5 activity attenuates excessive grooming and instrumental learning differentially, and rescues impaired striatal synaptic plasticity in Δe4-22(-/-) mice. These findings show that deficiency of Shank3 can impair mGluR5-Homer scaffolding, resulting in cortico-striatal circuit abnormalities that underlie deficits in learning and ASD-like behaviours. These data suggest causal links between genetic, molecular, and circuit mechanisms underlying the pathophysiology of ASDs